Lucigenin (LC++) is frequently used for the luminescent detection of superoxide. To detect superoxide, lucigenin must be reduced to the lucigenin cation radical (LCz+) which forms a dioxetane after reaction with superoxide. Spontaneous decomposition of the dioxetane intermediate gives rise to the chemilumunescence. In the present study we show, using the ESR spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO), that photoexcitation of lucigenin in the presence and absence of reducing agent NADH produces DEPMPO-superoxide and hydroxyl radical adducts. In the presence of SOD the signal due to DEPMPO-superoxide is not observed and only the ESR spectrum of the hydroxyl radical adduct is observed. The combination of SOD and catalase inhibits formation of both superoxide and hydroxyl adducts. Generation of superoxide was also demonstrated by cytochrome-c reduction which was partially inhibitable by SOD. The anaerobic photolysis of LC++ in the presence of reducing agent NADH leads to the formation of reduced lucigenin with spectral characteristics in the visible region. In the presence of air only NADH is consumed and there is no change in the absorbance of LC++. This probably implies that the photoexcitation of LC++ in the presence of NADH results in the formation of LCz+, which on interaction with oxygen regenerates LC++ and forms superoxide anion radical. Consequently, lucigenin cannot be used to measure superoxide formation in photobiology.